Wound-healing complications are an important health concern that can be associated with diabetes, bed sores, and infection. The inability to form a stable provisional matrix over the wound site is a common hallmark of poor wound healing. Without a stable matrix, the migration of inflammation responsive cells such as endothelial cells, neutrophils and macrophages, needed to produce new blood vessels1 and fight infections is not possible. The goal of this project is to develop an imaging method to quantify the stability of the provisional matrix during early-stage wound healing. This biological process has been largely invisible and the proposed work is expected to provide significant insight into molecular events that delay wound healing. Wound healing involves an intricate set of precisely timed processes that begin with the formation of a fibrin clot, followed by an inflammatory response, and eventually ending with extensive tissue remodeling and wound closure. During the inflammatory response, the provisional matrix is produced in part through the action of transglutaminase (TG) enzymes cross-linking plasma proteins such as fibrinogen and vitronectin. Recent work in our laboratory has shown that administration of infrared fluorescent-labeled substrates for the TGs, including both proteins and peptides, produces strong contrast for in vivo imaging.2 In this R21 project we will test whether our method can identify problems associated with unstable matrix formation in animal models by executing the following Specific Aims: (1) develop imaging strategies to support multiple time-point imaging in the healing cascade; (2) evaluate the performance of the multiple time point optical imaging method in vivo in a mouse model; and (3) compare provisional matrix stability through imaging matrix formation in a delayed wound healing model of the diabetic mouse. Multiple time-point imaging can monitor problems with both matrix formation and degradation processes. Slow matrix formation will be associated with poor incorporation of labeled substrate into the wound compared with controls. Excessive matrix degradation will be associated with rapid loss of labeled substrates in the wounds compared with controls. A successful project will lead to a new imaging method for studying wound healing and evaluating wound- healing response. In the near term, this method would be used in animal model studies to evaluate the wound- healing process and support the development of new approaches to promote wound healing. In the longer term, this method would be extended to clinical studies to identify the mechanisms of compromised healing and help in wound management. Furthermore, because similar cross-linking processes are involved in atherosclerosis3 and cancer,4 this imaging method will also find broader applications beyond wound healing.